1. Field of the Invention
The present invention is directed to a method for estimating the ratio of a target nucleic acid to the total amount of target nucleic acid and reference nucleic acid in the examined nucleic acid samples based on the amounts of amplification products in the reaction solutions at the endpoint of PCR reaction.
2. Description of the Related Art
Genetic examinations by analysis of nucleic acids are widely employed for medical, research, and industrial applications with recent progress in technologies of genetic manipulation, genetic recombination, and the like. These examinations are examinations that involve the detection and quantification of the presence of a target nucleic acid having a target nucleotide sequence in samples, and are applied in various fields, not only in the diagnoses and treatment of diseases, but also in examination of food. For example, genetic examinations for detecting congenital or acquired mutant genes, virus-related genes, and others are carried out for diagnosis of diseases, such as genetic diseases, tumors, and infections. Analysis of genetic polymorphisms, including single nucleotide polymorphism (SNP), is also applied not only to clinical examinations and academic research, but also to quality checks and traceability of foods and others.
Samples which are subjected to gene analysis are often of trace amounts, like specimens in clinical examinations. For this reason, genome fragments containing a target nucleic acid which is an objective of analyzation are usually amplified in advance by, for example, PCR (Polymerase Chain Reaction), and the amplified genome fragments are employed to detect and quantify the target nucleic acid. Methods for measuring the amount of a target nucleic acid in examined samples which is an objective to be analyzed, in general, involve preparing a series of samples containing the target nucleic acid at known concentrations, making a calibration curve by measuring the amounts of amplified products in the reaction solutions after the PCR reaction performed on this series, and determining, on the basis of this calibration curve, the amount of the target nucleic acid in the examined samples.
On the other hand, samples which are subjected to gene analysis are mostly living-body samples and thus are prone to being influenced by, for example, individual differences due to subjects from which the samples were taken, where and when the samples were taken, and methods for preparing and storing the samples. As a result, there are great variations among the samples and it is also difficult in many cases to compare the results of examinations. However, analysis can be made with high accuracy by using, as a reference nucleic acid, a nucleic acid which is relatively abundant in samples or a nucleic acid having a known content which can be expected beforehand, and determining the ratio between the amount of target nucleic acid and the amount of reference nucleic acid. For example, when the amount of target nucleic acid in two samples are compared, it is possible to ascribe the difference in the amounts of target nucleic acid in the two samples to significant difference between the sample, rather than sample variations between the samples, by comparing the ratio between the amount of target nucleic acid and the amount of reference nucleic acid in each of the samples, rather than comparing directly the amounts of target nucleic acid.
Methods for determining the ratio between the amount of target nucleic acid and the amount of reference nucleic acid in samples include, for example, competitive PCR processes in which at the endpoint of PCR, the amounts of amplification of target nucleic acid and of reference nucleic acid are compared and quantified to determine the ratio between the target nucleic acid and the reference nucleic acid. As a method for determining the ratio between the amount of target nucleic acid and the amount of reference nucleic acid in samples is disclosed, for example, (1) a method for determining the ratio between the types of nucleic acids in a group of nucleic acid samples, the method including the steps of amplifying in a gene amplification process mixtures of the respective nucleic acids in the group of nucleic acid samples; digesting the amplified products with a restriction enzyme; subjecting the digested products to electrophoresis; measuring the density of migrated bands; and determining the ratio between the types of nucleic acids in the group of nucleic acid samples based on the densities of the respective bands measured (see, for example, Japanese Patent No. 3,950,546). Also disclosed is (2) a method for quantifying a particular plant genus in foods or others, the method including preparing a correcting sample, in which a sample derived from the particular plant genus which is an objective to be detected and a sample of a standard plant are mixed at a predetermined ratio, followed by extracting genome DNAs from the correcting sample; preparing a sample to be examined, in which a known amount of the standard plant sample is added to a food or raw material of food which is an objective to be examined, followed by extracting genome DNAs from the sample to be examined; performing a real time PCR process with the genome DNAs and primers; and making a correction with a correcting reference value which is detected in the correcting sample, thereby determining by calculation the amount of raw material from the particular plant contained in the examined sample (see, for example, PCT International Publication No. WO 04/101794).
Competitive PCR processes are based on the assumption that the ratios between a reference nucleic acid and a target nucleic acid in the reaction solutions before the PCR reaction (initial template amount ratio) and after the PCR reaction (post-amplification template amount ratio) are almost equal, that is, that the reference nucleic acid and the target nucleic acid are amplified with the same efficiency. Thus, competitive PCR processes have a problem resulting in unreliable measured results when a reference nucleic acid and a target nucleic acid are amplified with different efficiencies.
PCR reactions, in principle, amplify a particular target nucleotide sequence by repeating 25 to 30 cycles, each cycle consisting of three steps: denaturation, annealing, and extension. At early reaction cycles, the target nucleic acid will be amplified 2n times after the cycle has been repeated n times and increased exponentially, whereas at the end of the reaction (endpoint), the PCR reaction will have become saturated. That is to say, the amounts of amplified product in the reaction solutions at the endpoint are not correlated with the amount of target nucleic acid at the beginning of the PCR reaction, and it is difficult to make a quantitative measurement. Therefore, the above-described method (1) in which the amplification product is detected and quantified in the reaction solutions at the endpoint has a problem of being incapable of making a measurement in the case where the PCR reaction is in saturation. On the other hand, the above-described method (2) employs real time PCR and thus does not pose a problem resulting from the saturation of the PCR reaction, but unlike common PCR, real time PCR has problems of requiring a more expensive apparatus and intricate considerations of reaction conditions.